Effect of Populus nigra spring and autumn leaves extract on Capsicum annuum infected with pepper mild mottle virus

Capsicum annuum is one of the main vegetable crops for the local market and exportation in Egypt. In this concern, pepper mild mottle virus (PMMoV) infection caused a significant decrease in Capsicum sp. leading to large economic losses. An isolate of PMMoV was obtained from naturally infected pepper plants, exhibiting different patterns of mottling, leaf distortion, yellowing, and stunting of leaves. The virus was identified. The molecular detection of PMMoV was done using RT-PCR with specific primers designed for coat protein genes. An RT-PCR product (474) bp of the coat protein gene of (PMMoV) was cloned. The target of the investigation was the effect of spring and autumn ethanol extracts of Populus nigra leaves on C. annuum seedling growth and infected C. annuum with (PMMoV) under greenhouse conditions. The experimental data showed that treated spring leaf extract of P. nigra enhanced infected C. annuum seedling growth parameters and fruit quality compared to uninfected seedlings. P. nigra spring leaf extract containing some allo-chemicals had a negative effect on uninfected seedlings. P. nigra autumn leaf extract significantly improved the growth and fruit quality of infected C. annuum seedlings compared to the control.

www.nature.com/scientificreports/ Pepper mild mottle virus (PMMoV) has only recently identified on commercial bell pepper fields in Florida, Italy, 13 . The virus had spread by mechanical means and infected seeds but cannot transmit by insects. It has grown worldwide in field-grown bell, hot, and ornamental peppers. It found in pepper cultivars where production practises are typical for the rapid spread of the disease 14 .
As foliar symptoms can be mild, infected plants may did not notice till fruit symptoms were evidently resulting in spread to adjoining plants and higher yield losses 13 . (PMMoV) causes serious economic losses in pepper production in China. 15 identified two PMMoV isolates (named PMMoV-ZJ1 and PMMoV-ZJ2) with decrement symptoms in a survey for viral diseases on pepper in Zhejiang province. (PMMoV) infected fruits general appeared small and malformed and this was obvious by off-colored sunken areas. 16 found that a virus was causing damage to pepper yield.
The main target of this demonstration evaluated leaves of Populus nigra as a natural woody trees products had a chemical values. In this concern applied to study the effect of foliar (25-50 and 100%) concentrations of spring and autumn leaves (APLE) on C. annuum seedlings and fruit parameters. As well as studying the effects of (25-50 and 100%) concentrations of (SPLE) and (APLE) foliar applications, the process took 20 and 40 days from germinating infected pepper under greenhouse conditions in the Ismailia region.

Material and methods
This investigated study highlight the important of Populus nigra leaves and indicator differences between chemical strictures during spring and autumn content. In this concern, clear new untraditional process struggle PMMoV infections. During the spring on 2020, the current trial had carried out at Ismailia Governorate, Egypt. After pepper seedlings had three or four true leaves transplanting, Capsicum annuum was cultivated on pots 30 cm in mixed loam and sand (1:1) under greenhouse conditions. Mineral fertilizer had using the nitrogen fertilizer in the structure of calcium nitrate (17% N), potassium fertilizers in the structure of potassium sulphate (48% K 2 O) and phosphorus fertilizers in the structure of phosphoric acid (61.5% P 2 O 5 ) according to Table 1.To investigate the effect of concentrations (% 25-50 and 100) foliar application of spring Populus nigra leaves extract (SPLE) and autumn Populus leaves extract (APLE) individuals after (20 and 40 days) respectively pepper transplant on growth parameters without infection as a split block treatment. On this concern pepper plant had infected with (PMMoV) during a first week after transplant, then treated at (20 and 40 days) respectively from transplant with foliar application extract, form (SPLE) and (APLE) individual as another split block treatment.
Greenhouse conditions. Greenhouse air was 21-30 °C during daylight and 16-18 °C at nighttime. Atmosphere had relative humidity from 40 to 90%. The suggested vapor pressure deficit (VPD) might be from 3 to 7 g/ m 317-20 . The solar radiation between 200 and 450 W/m 2 within the gable-even-span greenhouse was 6 measured and evidenced for short and long requisites. In contrast, other greenhouses conditions control using some apparatus, like black net sheets and natural airing systems 20 .
Tree materials. Collection of tree material poplar tree spring leaf collected in March 2019 and 2020 from trees growing in the nursery of timber trees department at the Horticulture Research Institute Agriculture Research Center, Giza, Egypt. Senescent leaves were collected in September (2018 and 2019). The samples dried in the electric oven at 40 °C until they reached constant weight, according to 21 . Dried material was ground by an electric mixer to find the crush forms of each sample. The powder preserved in sterilised glass jars.
Preparation extracts samples. Then samples were air dried in the laboratory for seven days under room conditions and later in the electric oven for two days at 40 °C 22 . The dried material then pulverised using a blender (electric mixer) to get powder forms of each sample. The powder collected and kept in clean and sterile conditions. Each leaf in spring and autumn, an individual dried powdered sample 500 g had lain in a 2000 ml beaker and processed by drenching 1000 ml of ethanol solvent. Then they enclosed with aluminium foil and put into a water bath 60 °C and had shaken to get homogenous solutions. After that, the samples filtered and  Fig. 2). The incidence of PMMoV confirmed by back inoculation with Chanopodium amaranticolor. The tested plants could divide, according to their reactions, into two groups:  Unsusceptible plants. These plant species were not susceptible to pepper infection. These plants belong to different families: Cucurbitaceae, Fabaceae, and N. arusica. Host range studies for diagnosis will usually be most useful for those infecting a relatively narrow range of plants 26 . The general outlook of the result in Table 2 indicator that the studied isolate of PMMoV had a wide host range between members of the family Solanaceae. On the other side, the virus infects a few species of Chenopodiaceae. PMMoV induced mottling, yellowing, and malformation symptoms in the family Solanaceae. The informed data in Table 1 confirmed the results of 27 .

Modes of transmission. Mechanical transmission. Inoculums prepared by homogenising infected pep-
per leaves with a few drops of phosphate buffer (pH 7.2) in a sterilised mortar. Leaves of host plants previously dusted with carborundum (600 mech) rubbed with the forefinger or with a cheesecloth pad previously soaked in the inoculum. The plants rinsed with tap water and kept in the insect proof greenhouse. Obtainment results revealed that PMMoV easily transmitted mechanically to indicator hosts like Chenopodium amaranticolor which indicator chlorotic local lesions.
Insect transmission. Two aphid species, name, Aphis faba (scop) and Myzus persicae (sulz) checked for their ability to transmit the isolated virus. A. faba (scop) and M. persicae (sulz) maintained on virus-free health faba beans for A. faba (scop) and cabbage plants for M. persicae (sulz) and kept under insect-proof cages in the greenhouse. The aphids starved for one hour and then transferred to feeding for a 30 min acquisition feeding period on diseased pepper plants. At the end of the feeding period, aphids transferred to healthy plants at a rate of 10 aphids/plant. After a 24 h feeding period, the insects had killed by spraying all tested plants with an effective insecticide (malathion 0.2%). Symptoms and the percentage of transmission recorded.
Results indicator these A. faba (scop) and M. persicae (sulz) did not able to transmit the virus. None of the tested plants produced any symptoms.
Gas chromatography (GC) analysis. Varian 3400 chromatography line, 30 cm in height and 0.32 mm in width, was working with helium as a transporter gas. GC temperature software program Spectra of mass saved in electron ionization (EI) form at 70 eV. The check repetition ranged over a mass of atomic mass units.
Statistical design and analysis. The design was a completely randomized block (RCBD) with five replicates. For each treatment, the least significant differences (LSD) were used to test the differences among the means of each parameter.

Results
Populus nigra leaf extract chemical composition in autumn and spring. The investigated data indicator that there were differences in the chemical structure of P. nigra extract between spring and autumn periods under Egypt conditions. It had clear that spring (PLE) extract more enhanced the chemical composition present compared to the autumn extract period. In comparison the autumn (PLE) extract, the spring (PLE) extract contained more tiglic acid, phenol, benzoic acid, dihydrocinnamic acid, cinnamic acid, 4-hydroxyphenylacetic acid, 4-methoxycinnamic acid, 3,4-dimethoxymethyl cinnamate, ferulic acid, caffeic acid, and pinostrobin chalcone as shown on Table 3.  www.nature.com/scientificreports/ control while (PMMoV) infected pepper (PMMoV + PLE25 and PMMoV + PLE50%) more significant capable than (PMMoV + PLE100). This side P. nigra spring leaf extracts 25% concentration application on health pepper more significantly enhanced than poplar leaves application 50% on pepper number branches as well as (PMMoV + PLE25) the highest significant mean value compared to other infected pepper plants. Fruit weight of pepper plants data had indicator that treated with (PLE 100 and PMMoV + PLE50%) had no significantly mean value between (healthy and infected pepper, respectively. PLE 25 and PMMoV + PLE25 recorded the high Table 3. Chemical composition of P. nigra leaves extract during autumn and spring.

Effect of (PLE) after 20 and 40 days from (PMMoV) on symptoms. Using (SPLE) foliar application
with different concentrations, processed infected pepper plants enhanced fruit pepper virus symptoms compared to untreated infected fruit peppers, a regard to Table 6. According to data shown in Table 7, treated infected peppers with 50 and 100 APLE concentrations were more capable of battling virus symptoms of pepper infected peppers when compared to unprocessed infected peppers. Meanwhile, foliar application with (25% APLE is incapable of managing symptoms of (PMMoV). Table 8 indicated that treated infected pepper with spring (PLE) foliar application enhanced pepper defense for (PMMoV) compared to untreated. By using (PLE100%) recorded the highest significant decrease in treatment after the first and second. The second season had the same trend. On the other hand, autumn (PLE) applications lead to significantly decreased virus concentration and reproductive.

Discussion
Populus leaves turn yellow in autumn, a consequence according to chlorophyll degradation during senescence in response to environmental change 29,30 . The present study showed that concentrations of phenolic acids in leaves were more highly elevated at the beginning of the spring period and decomposed at the end of the active season until leaves maturity 31 . 32 found that willow trees' degraded structure and growth rate turn down on senescence leaves in relation to low leaf water potential. Senescence process leaf damage caused by uncoupled chlorophyll that downstream decreased led to the photosynthesis process not operating and increased chemical decomposition in 33,34 . This concern 75.0% diluted ethanol solvent had a significant effect in extracting phenol composition 35 .
In fact, chlorophyll loss might be a sign of membrane damage, especially if hydrogen peroxide generation is enhanced 36 . By destroying a variety of targets, including proteins, reactive oxygen species can cause significant harm to cell structure and metabolism 37 . In fact, chlorophyll loss might be a sign of membrane damage, especially if hydrogen peroxide generation is enhanced 36 . Reactive oxygen species have the potential to cause significant cell damage. Indeed, phenolic compounds appeared to reduce seedling growth of crop 38 . Meanwhile, phenol structures caught hold of the behaviour of respiratory enzymes. Typically pretentious phenol components are aldolase plus glucose phosphate isomerase, involved in glycolysis and glucose 6-phosphate dehydrogenase 39 .
On the other side, Populus is a genus of the Fam. Salicaceae, and included different naturally happening aromatic components such as salicylic acid and salicylic alcohol, as well as aromatic ketones moreover terpenoids furthermore fatty addition to organic acids plus benzyl alcohol also beta-phenyl ethanol, moreover other compounds 40,41 . The researchers also recorded that the majority structures had showed antimicrobial motion, such as salicylic component 42 .
The study present that aspen had different concentrations of deterrent secondary substances relating to bud grow old. This concerning four herbivores had incapable feed selective as defense protection [43][44][45] . Poplar trees had a surprising occurrence famous as autumn senescence, a vital role in survival trees 46 . The commencement   www.nature.com/scientificreports/ of inception senescence had known altering in metabolic rate of leaf since starting copious photosynthetically active to senescence situation then leaf actively had been incapable of precious components content after that transported out leaf 47,48 . Senescence leaves had more than greater synchronized development in cell organs. It had deconstructed and content reallocated 49 . While the tree cell gets the beginning pointer for preparatory programmed death. Cell dismantled itself then manner initial chlorophyll molecules after that nucleus and mitochondria dismantled 50 . 51 indicator that the exogenous function of salicylic acid enhanced the photosynthetic 52 observed the encouraging consequence of low salicylic acid concentration on increased yield. When cucumber and tomato plants treated with reduced salicylic acid concentrations, fruit output parameters enhanced considerably 53 .
On this concern, 54 found that foliar application of salicylic acid had a positive effect on early yield and total yield, and that the highest yield occurred in the 0.50 mM salicylic acid treatment. They also suggested that to improve yield, foliar application of salicylic acid had used. On this mention low doses, salicylic acid is more capable of photosynthesis and growth parameters than excessive amounts 55 .
Higher salicylic acid concentrations [10-4 M] inhibited ethylene synthesis, according to 56 . However, the mechanism of action of salicylic acid-mediated ethylene biosynthesis is still unknown. The data had pinpointing the mechanism connected with salicylic acid for ethylene biosynthesis and action will require a lot of debate. 57 found chlorophyll reduced following concentrations (100-1 mM) of salicylic acid submission in leaves 58 reported that virus ability to inhibit or enhance according to salicylic acid dependent signaling.
Salicylic acids enhance the confrontation mechanisms of phytoalexin construction then being capable of cell wall membrane amplification and lignification, furthermore salicylic acid submission on tobacco improved the confrontation with viruses and the resistance movement among plant cells 59 . 60 investigated that salicylic acid, an important component, had encouraged endogenous messengers which enhance pathogen resistance. This theory has maintained tobacco plants' foliar application by salicylic acid, and this improvement persuaded structures of pathogenesis-related proteins and confrontation with the tobacco mosaic virus 61 . This admiration 62 reported that instruction of virus resistance with phenol stricture is possible. 63 found that salicylic acid phytohormone participants in modifiable protection were mostly against abiotrophic moreover hemotrophic pathogens.
The preceding study observed that salicylic acid reduced virus concentrations detected by DAS-ELISA. The diminution in virus concentrations released an enhanced peroxidase enzyme, which is recognized to encourage forming polymerization. It lead to lignin combination plus point had straight connected with an augmented facility of systemically secluded lignin tissues furthermore assist protection from viral infection 64 . This concern salicylic acid excesses production of antioxidants moreover improved virus resistance. Accumulation of salicylic acid following the process had induced in plants challenged by various viruses 65 . An improvement in salicylic acid concentration was essential for plants' resistance to viral infection and virus replication 65 .

Conclusion
The experimental data expresses the new applied method using Populus nigra leaves extract to increase poplar trees evaluation. Pepper is Egyptian economic and popular crop. Pepper mild mottle virus damaged pepper crops without infect side. The other side Populus are deciduous trees and suitable for Egyptian conditions. Ethanol extract of Populus leaves analysis recorded high antimicrobial content during spring season. Meanwhile, autumn Populus leaves analysis chemical contented before falling. This investigated study effect of different concentrations (25-50 and 100%) as foliar applications of spring and autumn Populus leaves individuals on growth parameters of healthy and virus infected pepper. Populus leaves extracts foliar applications treated after (20 and 40 days) from pepper transplants. The experimented data recorded spring Populus leaves extract had a (negative and positive) significant effect on health and infected pepper growth, respectively on high concentrations. On the other side, autumn foliar Populus leaves extracts high concentrations had significant increment on pepper growth and fruits.

Data availability
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.